DFNB3 mutations result in recessive nonsyndromic profound congenital hearing impairment. The map position of DFNB3 was refined to 17p11.2 (Liang et al. 1998). The goal of this project during the past year was to positionally clone DFNB3 and understand its essential biological function in the auditory system. To this end we have: (1) identified two families from India with congenital recessive hearing impairment due to mutations in DFNB3; (2) refined the map position of DFNB3 to 17p11.2; (3) functionally cloned the homologue of DFNB3 (shaker2, congenitally deaf mouse strain); (4) sequenced a BAC that rescued the shaker2 phenotype; (5) identified the DFNB3 and shaker2 genes as the same novel unconventional myosin gene; and (6) characterized the myosin15 gene from both the mouse and human. Myosin-15 represents a new branch of the unconventional myosin family. The function of this gene product in the auditory system is unknown. The genomic and cDNA structures of the human (MYO15) and mouse (Myo15) myosin-15 genes have now been elucidated. The full-length cDNAs are ~ 8 kb and contain open reading frames derived from 63 exons. The encoded proteins are 87 % similar and have predicted molecular masses of ~ 260 kDa. Analysis of RT-PCR products indicates that additional myosin-15 isoforms may result from alternative splicing. Ongoing expression studies suggest that myosin-15 transcription may be restricted to relatively few cell types. By using RT-PCR, myosin-15 transcripts were detected in the inner ear in both humans and mice. In mice, this finding was confirmed and extended by in situ hybridization studies which revealed Myo15 transcripts in cells of the developing cochlea and vestibular organ. As an additional and important tool to study myosin-15 expression and function, rabbit polyclonal anti-Myo15 antibodies have been raised against two synthetic and two recombinant peptides. Preliminary analysis indicates that antibody production against all of the peptides was successful. In collaboration with Dr. Bechara Kachar, the Myo15 antibodies will also be used to examine the ultra-structural location of Myo15 in the inner ear at the level of electron microscopy.